The prominent risk factor for Alzheimer's Disease (AD) is aging. Animal studies establish an intimate link between signaling pathways that regulate lifespan and processes that control cellular protein homeostasis, including insulin growth factor 1 receptor (DAF-2/IGF-1R) signaling, dietary restriction (DR) and mitochondria! electron transport chain perturbations. The overall goal of Project 2 is to study cellular aging or senescence in cell-based models that enable us to understand more clearly how aging affects the cell biology that can lead to loss of protein homeostasis control and the onset of AD. In Aim 1, we hypothesize that aging fundamentally changes the processing of APP into Abeta. We will utilize established fibroblast cell-based aging models and we will develop neuronal cell-based aging models to first demonstrate that aging signaling pathways that control the lifespan of worms and mice also influence cellular lifespan in culture. We will then express APP in these cell-based aging models to discern whether the youthful control of protein homeostasis is lost upon aging of the cells and whether DAF-2/IGF-1R signaling and related pathways influencing aging will extend their lifespan and protect the cells from proteotoxicity, as has been observed in whole animal models including worms and mice. Biochemical and morphological approaches including western blotting, immunofluorescence (IF) and immunoelectron microscopy (IEM) will be used to follow the processing of APP into Abeta, the subcellular appearance of aggregates and their spatial and temporal correlation with proteotoxicity contributing to senescence and cell death. In Aim 2 we will utilize the aging models to study the membrane trafficking pathways to explore the hypothesis that they function well in young cells yet appear to fail in old cells, in an effort to begin to understand how the expression of APP and its conversion into Abeta becomes toxic with aging in the context of its extensive intracellular processing and trafficking. Aim 2 is distinguished from Aim 1 by the utilization of numerous known biological perturbants of membrane trafficking pathways in a systematic fashion to discern how these pathways influence the processing of APP into Abeta the subcellular appearance of aggregates and their spatial and temporal correlation with proteotoxicity in the context of aging.